Identification of the unknown transformation products derived from clarithromycin and carbamazepine using liquid chromatography/high-resolution mass spectrometry.

RATIONALE: A comprehensive study of the environmental fate of pollutants is more and more required, above all on new contaminants, i.e. pharmaceuticals. As high-resolution mass spectrometry (HRMS(n)) may be a suitable analytical approach for characterization of unknown compounds, its performance was evaluated in this study. METHODS: The analyses were carried out using liquid chromatography (LC) (electrospray ionization (ESI) in positive mode) coupled with a LTQ-Orbitrap analyzer. High-resolution mass spectrometry was employed to assess the evolution of the drug transformation processes over time; accurate masses of protonated molecular ions and sequential product ions were reported with an error below 5 millimass units, which guarantee the correct assignment of their molecular formula in all cases, while their MS(2) and MS(3) spectra showed several structurally diagnostic ions that allowed characterization of the different transformation products (TPs) and to distinguish the isobaric species. RESULTS: The simulation of phototransformation occurring in the aquatic environment and identification of biotic and abiotic transformation products of the two pharmaceuticals were carried out in heterogeneous photocatalysis using titanium dioxide, aimed to recreate conditions similar to those found in the environmental samples. Twenty-eight main species were identified after carbamazepine transformation and twenty-nine for clarithromycin. CONCLUSIONS: This study demonstrates that HRMS, combined with LC, is a technique able to play a key role in the evaluation of the environmental fate of pollutants and allows elucidation of the transformation pathways followed by the two drugs.

Fate of antibacterial spiramycin in river waters.

Spiramycin, a widely used veterinary macrolide antibiotic, was found at traceable levels (nanograms per litre range) in Po River water (N-Italy). The aqueous environmental fate of this antibiotic compound was studied through drug decomposition, the identification of the main and secondary transformation products (TPs), assessment of mineralisation and the investigation of drug TPs toxicity. Initially, laboratory experiments were performed, with the aim of stimulating the antibacterial transformation processes followed in aquatic systems. The TPs were identified through the employment of the liquid chromatography (LC)-mass spectrometry technique. Under illumination, spiramycin degraded rapidly and transformed into numerous organic (intermediate) compounds, of which 11 could be identified, formed through five initial transformation routes. These laboratory simulation experiments were verified in situ to check the mechanism previously supposed. Po River water was sampled and analysed (by LC-high-resolution mass spectrometry) at eight sampling points. Among the previously identified TPs, five of them were also found in the river water. Three of them seem to be formed through a direct photolysis process, while the other two are formed through indirect photolysis processes mediated by natural photo sensitisers. The transformation occurring in the aquatic system involved hydroxylation, demethylation and the detachment of forosamine or mycarose sugars. Toxicity assays using Vibrio fischeri proved that even if spiramycin did not exhibit toxicity, its transformation proceeded through the formation of toxic products.

Study of the photolytic and photocatalytic transformation of amiloride in water.

The diffusion of drug residues in wastewaters and surface waters as rivers and streams may constitute a problem for the environment, with consequences on the ecosystem and also on the human health. This paper deals with the study of the photo-induced transformation of amiloride, an orally administered diuretic agent, under simulated solar light. Direct photolysis and photocatalyzed degradation processes, using titanium dioxide as a photocatalyst, were investigated. The study involved the monitoring of the drug decomposition, the identification of intermediate compounds of the decomposition, the assessment of mineralization, as well as the evaluation of the toxicity associated to the degradation products. Amiloride underwent complete degradation within 30min of irradiation (heterogeneous photocatalysis) or 4h (homogeneous photolysis). HPLC coupled to HRMS, via ESI interface, demonstrated to be a powerful tool to identify and measure degradation products of the studied drug. By considering the photocatalytic process, the identified intermediates are formed through: (1) dechlorination and hydroxylation of the heteroaromatic ring; (2) the detachment of the guanidinic moiety; (3) cleavage of the heteroaromatic ring. The drug photomineralization was a rather slow process and after 4h of irradiation 25% of the total organic carbon (TOC) was still present. Chlorine was stoichiometrically released as chloride ions within the considered irradiation times (4h), while nitrogen was only partially converted into ammonium ions. This was due to the formation of guanidine, known to be hardly mineralized photocatalytically, and some other small molecules still containing the nitrogen. Acute toxicity, measured with a Vibrio fischery assay, showed that amiloride transformation proceeded through the formation of toxic compounds.

Mass spectrometry analysis of IgA1 hinge region in patients with IgA nephropathy.

BACKGROUND: Physicochemical alterations of the IgA molecule are supposed to play a pathogenetic role in IgA nephropathy (IgAN). The present study was carried out to analyze the structural variety of O-glycans on the IgA1 hinge region in IgAN. Sera from 9 IgAN patients and 9 healthy controls were individually examined to evaluate the IgA1 content and binding lectins (jacalin and Helix aspersa), using enzyme-linked immunosorbent assay (ELISA) techniques. The IgA1 from pooled sera were separated by affinity chromatography (jacalin), and the fragment containing the hinge region was prepared by pyridylethylation and trypsin treatment. The IgA fragments containing the hinge glycopeptide (33-mer hinge peptide core (HP) + O-glycans) were separated by jacalin affinity chromatography. Because we used jacalin, we only analyzed the Gal-3GalNAc residue containing IgA. The molecular weight (MW) of the IgA1 fragments was estimated using an ion trap mass spectrometer equipped with an electrospray ion source (ESI/MS). RESULTS: IgA1 concentration in pathological sera was higher than in the control serum (p<0.01). Compared with controls, serum IgA1 from IgAN patients showed significantly greater binding to the 2 lectins, jacalin (p<0.01) and Helix aspersa (HA, p<0.001), which are specific for O-linked Gal-beta1,3-GalNAc and GalNAc, respectively. Analyses of pooled sera showed that the number of O-glycosidic chains was comparable in IgAN and normal sera. With regards to the individual residues, we found that IgAN sera contained less sugar and galactose and sialic acid moieties than sera from control subjects, was reduced in IgAN sera, while terminal N-acetylgalactosamine levels were higher when compared with normal serum. CONCLUSIONS: Abnormalities of hinge region O-linked glycans were confirmed using advanced spectrometry technology. The pathogenetic implications for aggregation and defective removal of IgA1 are discussed.

Synergistic action of ADP and 2,3-bisphosphoglycerate on the modulation of cytosolic 5'-nucleotidase.

Cytosolic 5'-nucleotidase, acting preferentially on IMP, GMP and their deoxyderivatives, can also behave as a phosphotransferase, operating a transfer of phosphate from a nucleoside monophosphate donor to a nucleoside acceptor which, besides a natural nucleoside, can be also an analog. The enzyme activity is stimulated by ADP, ATP and 2,3-bisphosphoglycerate (BPG). The concentration of effector required to attain half maximal activation (A0.5) for the bisphosphorylated compound is in the millimolar range, so that BPG seems to act as a physiological activator of 5'-nucleotidase only in erythrocytes. However, the combination of BPG and ADP brings about a significant increase of their respective affinity for the enzyme, lowering their A0.5 values approx. 4-times. The observation that BPG favors the phosphotransferase more than the hydrolase activity of 5'-nucleotidase stands for a key role of this metabolite in the regulation of the processes of activation of purine pro-drugs, in which this enzyme is involved.

Application of semipermeable membrane device for assessing toxicity in drinking water.

Semipermeable membrane devices (SPMDs) mimic passive diffusive transport of bioavailable hydrophobic organic compounds through biological membranes and their partitioning between lipids and environmental levels. Our study was developed on a surface water treatment plant based in Turin, Northern Italy. The investigated plant treats Po River surface water and it supplies about 20% of the drinking water required by Turin city (about one million inhabitants). Surface water (input) and drinking water (output) were monitored with SPMDs from October 2001 to January 2004, over a period of 30 days. The contaminant residues, monthly extracted from SPMDs by dialysis in organic solvent, were tested with the Microtox acute toxic test and with the Ames mutagenicity test. Same extracts were also analyzed with gas chromatography--mass spectrometry technique in order to characterise the organic pollutants sampled, especially Polycyclic Aromatic Hydrocarbons (PAHs). Although the PAHs mean concentration is about one hundred times lower in the output samples, the mean toxic units are similar in drinking and surface water. Our data indicate that the SPMD is a suitable tool to assess the possible toxicity in drinking water.

Polycyclic aromatic hydrocarbons degradation by composting in a soot-contaminated alkaline soil.

This study deals with the biodegradation of the polycyclic aromatic hydrocarbons (PAH)s present in a soil contaminated by soot waste, characterised by a total PAHs content in the 200 mg kg(-1) range. A challenging characteristic of the waste soil treated was its high alkalinity, with a pH of about 12.8. The waste came from a soot-contaminated area located in the industrial zone of Porto Marghera, Venice (Italy). The biodegradation process employed was the composting of the waste with sewage sludge and yard waste. The process was carried out on a pilot scale using a closed tank with forced aeration for a period of 60 days, followed by 70 days with natural aeration. The time evolution of the process was monitored by following the time change in the concentration of the 16 US-EPA PAHs, as well as temperature, pH, electrical conductivity, C and N contents. Also phytotoxicity parameters, such as the growth and respiration indexes, were monitored. An induction time of about 30 days was observed, which corresponded to the time required before observing a significant self-drop in the waste pH and an increase in mass temperature. Afterward, a progressive drop in the PAHs concentration was observed, up to reaching after 130 days an overall degradation percentage in the order of 68%. The degradation was more effective on rather low molecular weight PAHs (2-4 rings).

The phosphotransferase activity of cytosolic 5'-nucleotidase; a purine analog phosphorylating enzyme.

Cytosolic 5'-nucleotidase is involved in the phosphorylation of several purine nucleoside analogs,used as antiviral and chemotherapeutic agents. In order to assess its role in the mechanisms of activation and inactivation of purine prodrugs, it is essential to study the regulation of both hydrolase and phosphotransferase activities of the enzyme. Using a zone capillary electrophoresis apparatus, we were able to separate substrates and products of the reactions catalyzed by cytosolic 5'-nucleotidase. The method overcomes the frequent unavailability of radiolabeled substrates, and allows the influence of possible effectors and/or experimental conditions on both enzyme activities to be evaluated simultaneously. Results showed that the enzyme was able to phosphorylate several nucleosides and nucleoside analogs with the following efficiency: inosine and 2'-deoxyinosine > 2',3'-dideoxyinosine > 6-chloropurineriboside > 6-hydroxylaminepurine riboside> 2,6-diaminopurine riboside > adenosine > cytidine > deoxycoformycin > 2'deoxyadenosine. This is the first report of deoxycoformycin phosphorylation catalyzed by a 5'-nucleotidase purified from eukaryotic cells. The optimum pH for nucleoside monophosphate hydrolysis was 6.5, slightly more acidic than the optimum pH for the transfer of the phosphate, which was 7.2. Finally, the presence of a suitable substrate for the phosphotransferase activity of cytosolic 5'-nucleotidase caused a stimulation of the rate of formation of the nucleoside. The results suggest the requirements for phosphorylation of nucleoside analogs are a purine ring and the presence of an electronegative group in the 6 position. The stimulation of the rate of nucleoside monophosphate disappearance exerted by the phosphate acceptor suggests that the hydrolysis of the phosphoenzyme intermediate is the rate-limiting step of the process.

Treatment of advanced renal cell cancer with sequential intravenous recombinant interleukin-2 and subcutaneous alpha-interferon.

Starting from in vitro studies suggesting synergistic antitumour activity against renal cell cancer (RCC) of recombinant interleukin-2 (rIL-2) and alpha-interferon (IFN), a phase II trial was initiated to test the clinical activity of this combination. The two cytokines were administered sequentially, with the aim of reducing the risk of additive toxicity and enhancing the immunological reaction against the tumour. The original treatment schedule consisted of rIL-2 18 x 10(6) U/m2/day by continuous intravenous infusion for 120 h days 1-5, and alpha-IFN 2b, at a flat dose of 9 x 10(6) U by subcutaneous or intramuscular injection thrice in a week, from day 8 to 28. Treatment was planned to be continued for six or more 28-day cycles, depending on clinical response. 12 patients were treated according to this schedule; as some cardiovascular toxicity was experienced in this set of patients, 11 further patients were treated with half-dose rIL-2 (i.e. 9 x 10(6) U/m2/day). 17 out of 23 enrolled patients completed at least one cycle of treatment and were evaluated for response. We observed six major responses [one complete response (CR) + five partial responses (PR)] for an objective response rate of 35% [95% confidence interval (CI) 17-59%]. 5 additional patients achieved stabilisation of disease; one of them reached CR after surgical extirpation of a lung mass. Sites of response included lung, nodes and bone. Duration of response is 12+ months for CR; 17, 16, 12+, 9 and 9 months for PRs. Median survival is 16 months. Response was not significantly different between full-dose and half-dose rIL-2. Considering stable disease (SD) as responses, there seemed to be a higher chance of response for patients with smaller tumour burden (P = 0.032). The toxicity of rIL-2 treatment, mainly cardiovascular, was substantial; 9 patients experienced severe cardiotoxicity, consisting of major arrhythmias, myocardial ischaemia, reduction of ejection fraction measured with heart radionuclide scan, and were excluded from continuing treatment. Other rIL-2-related toxicities forcing exclusion from the study were severe thrombocytopenia (1 case), and generalised exfoliative dermatitis requiring steroids (1 case). Otherwise, treatment was well tolerated; rIL-2-related toxicities promptly recovered after rIL-2 discontinuation in the majority of cases, and no treatment-related deaths were reported. The half-dose rIL-2 regimen was significantly less toxic in terms of hypotension (P = 0.014), fever (P = 0.014), oliguria (P = 0.042), serum creatinine elevation (P = 0.009) and prothrombin time elongation (P = 0.038).(ABSTRACT TRUNCATED AT 400 WORDS)

Incidence of risk of thromboembolism during treatment high-grade gliomas: a prospective study.

A prospective study of a series of 77 patients on adjuvant radiochemotherapy following surgery for high-grade gliomas was conducted to evaluate the risk of deep vein thrombosis and identify risk factors. We found a 20.8% risk of deep vein thrombosis at 12 months (standard error = 4.8%) and a 31.7% risk (standard error = 7.4%) at 24 months (Kaplan-Meier method). Twenty patients (26%) developed deep vein thrombosis with a maximum incidence within the first 7 months after surgery when chemotherapy was still being administered, often with corticosteroids. The risk factors identified were histology (glioblastoma versus anaplastic astrocytoma, P = 0.032, log rank test; 0.0485 L-ratio) and the presence of paresis (P = 0.010, log rank test; 0.0161 L-ratio). A borderline tendency was found for an association between the deep vein thrombosis site and the side of paresis (P = 0.103, Fisher's exact test). Four patients (5%) had massive pulmonary embolism, which was fatal in 3 (4%).

Ion trap tandem mass spectrometry study of dexamethasone transformation products on light activated TiO2 surface.

The photocatalytic transformation of dexamethasone and the formation of its intermediate compounds have been studied using titanium dioxide as a photocatalyst. The degradation of dexamethasone occurs easily through the formation of several hydroxy derivatives whose characterization has been made by HPLC/MS/MS. Even if both oxidative and reductive processes can be operating, only oxidative products have been identified in air saturated aqueous suspensions. A pattern of reaction pathways accounting for the observed intermediates is proposed. The obtained experimental evidence may be rationalized postulating the existence of a double initial mechanism. A single oxidation step resulting from the attack by one *OH radical leading to the formation of five hydroxy-derivatives and a concomitant attack involving two *OH radicals leading to the hydroxylation of the quinoid moiety of the molecule.

Photodynamic therapy in gynaecological neoplastic diseases.

Since 1982, our department has used photodynamic therapy (PDT) in the treatment of loco-regional recurrences of gynaecological cancers. We have treated 26 patients in this time. In the majority of cases the site of vaginal recurrences was the vaginal vault. The light sources were an Argon-dye laser (Meditec) and, in some cases, a CO2 laser. The light dose ranged between 60 and 500 J cm-2. The photosensitizing drug used was Hematoporphyrin (HP) (Monico Farmaceutici) at the dose of 5 mg kg-1 body weight. Patients were evaluated 45 days after the treatment with a gynaecological examination and after 75-90 days with a vaginal smear. The results were divided into 2 groups: objective and symptomatic. The symptomatic response concerned only the patients treated with a palliative aim and, in this case, a complete response (CR) was a complete absence of symptoms at least for 60 days. In this group the complete response rate was 66%. In the curative group, the complete response was a cytological and/or histological absence of lesions. In this group we had 12 CR (70.58%). The survival rate was also evaluated. A significant review of the photodynamic therapy in gynaecological neoplasms has also been done.

Definitive evidence for the actual contribution of yeast in the transformation of neutral precursors of grape aromas.

Experiments were designed to demonstrate the actual contribution of yeast in the formation of the primary aroma during the vinification of neutral grapes. Ruché was chosen as the model wine to study because of its unique fragrance. A yeast strain specific for Ruché was selected using a new and rapid isolation method for red wines. The results of this study can be summarized as follows: Skins from nonaromatic white or red grapes apparently contain most of the primary aroma compounds that are revealed in the must only after contact with yeast cells under defined conditions. Similar results were obtained with the pulp and seeds fractions; however, the olfactory notes, although well characterized, differed from those obtained with skins alone. Clarification, filtration, and centrifugation of the pulp and seed fractions or sonification of the skins produce different and well-characterized olfaction notes during the contact with yeast. The primary aroma of nonaromatic white and red grapes contained in the skins can be revealed within 24-48 h of yeast contact in a synthetic nutrient medium (SNM). The primary aroma precursors extracted from the skins with methanol, water-saturated butanol, or aqueous buffer at pH 3.2, concentrated and eluted from a C18 resin column, can be transformed to the free form wine aroma markers within 6 h of contact with yeast cells in SNM. By contrast, prolonged maceration of the skins in aqueous alcoholic buffer at pH 3.2 or 1.1, at 50 or 70 degrees C did not release primary odors typical of wine. The individual primary aroma compounds, identified by GC-MS analysis in Ruché wine samples or in Ruché skin-yeast-SNM samples, could not explain the complexity of the typical Ruché wine odor. Only odors common to many wine varieties were identified by GC-olfactometry analysis.

Reversed-phase HPLC separation of complex mixtures of trace metals as dibenzyldithiocarbamate chelates.

The dibenzyldithiocarbamate chelates of Cd(II), Pb(II), Bi(III), Hg(II), Ni(II), Cu(II), As(III), Fe(III), Co(III) and In(III) are separated by reversed-phase HPLC in isocratic conditions. The procedure is simple, rapid, and gives satisfactory separations with high efficiency and sensitivity at mobile phase compositions very rich in organic modifier (85-88% CH(3)CN). The detection limits range from 1.4 to 14 mug/1. The elution order is correlated with the ability of the central metal atom to affect the electronic distribution of the ligand, which has readily polarizable donor atoms. Infrared spectroscopy data corroborate this assumption.

Use of methyliminodiacetic acid bound to cellulose for preconcentration and determination of trace-metal cations.

Methyliminodiacetic acid immobilized on a cellulose support can be conveniently utilized for trace-metal uptake. Capacities and uptake yields as a function of pH have been evaluated for Pb(II), Cu(II), Mg(II), Ca(II), Cd(II), Zn(II), Co(II), Ni(II) and Hg(II) and the results compared with those predicted from the stability constants for the same systems in homogeneous solution. The immobilization of the ligand increases its co-ordinating ability. The applicability to speciation studies is considered.

Retrospective analysis of 156 cases of metastatic renal cell carcinoma: evaluation of prognostic factors and response to different treatments.

BACKGROUND: Metastatic renal cell carcinoma is a "capricious" tumor. Many prognostic factors have been evaluated, treatment is still controversial, and results are not coincident. METHODS: We reviewed 156 patients with metastatic renal cell carcinoma. Survival from the time of diagnosis was the end point of the study. The influence on survival of age, sex, nephrectomy, disease-free interval, performance status, site and number of metastases was analyzed. Univariate and multivariate analysis were done. Survival according to different therapies was also evaluated. RESULTS: In our study, no nephrectomy, a disease-free interval < 24 months, > 2 metastatic sites and a performance status > 2 proved to be risk factors. According to the number of risk factors, 3 groups of patients were identified (low, intermediate and high risk). We observed 3 kinds of responses to treatments: 1) in untreated patients (n = 48), median overall survival was 6 months, and the 24-month survival rate was 8%; 2) in patients treated with hormone therapy and/or chemotherapy (n = 73), median overall survival was 13 months, and the 24-month survival rate was 24%; 3) in patients treated with interferon and/or interleukin-2 (n = 35), median overall survival was 16 months and the 24-month survival rate was 34%. CONCLUSIONS: Our results are only partially in accordance with those observed by other authors. Risk factors and treatment must be determined in more defined and selected studies.

Continuous non chronomodulated infusion of floxuridine in metastatic renal cell carcinoma (MRCC): report of 17 cases.

AIMS AND BACKGROUND: MRCC responds poorly to usual treatments. Recently floxuridine (FUDR) has been administered by chronomodulated infusion, obtaining interesting results. In order to simplify the infusion, we used continuous non chronomodulated infusion. METHODS: We treated 17 patients affected by MRCC with continuous non chronomodulated infusion of FUDR. Toxicity was evaluated according to WHO criteria. Responses were recorded as complete response (CR), partial response (PR), stable disease (SD) and progressive disease (PD). RESULTS: Sixty-four courses of therapy were administered; 15/17 patients, treated with a median of 4 cycles, were evaluable for the response. Only 1 patient showed a grade 3 toxicity (mucositis and diarrhoea); 6 patients showed grade 1-2 diarrhoea; 2 grade 1-2 nausea and vomiting; 1 grade 2 anaemia and thrombocytopenia. No patient obtained CR; 2 PR (lasting 7 and 9 months respectively) and 4 SD (lasting 4,5,6 and 9 months) were observed. CONCLUSIONS: In our experience continuous non chronomodulated infusion of FUDR did not show important general toxicity. The observed responses were not good enough. We think that a better selection of patients (good performance status) and the use of FUDR in an earlier stage of disease, can obtain better results.

Pancreatic cystadenocarcinoma and pregnancy: a case report.

We report a case of cystadenocarcinoma occurring in a pregnant woman. After child birth, a subtotal pancreatectomy was performed, without rupture of the cyst. The patient is asymptomatic, 24 months after surgery. The presentation of cystadenocarcinoma in pregnancy has been reported in another single case. The possibility of hormonal dependence is discussed.

Possible correlation between some biologic effects and the clinical course in patients treated with continuous infusion of interleukin-2 plus alpha-2 interferon for metastatic renal cell carcinoma.

BACKGROUND: Interleukin-2 therapy is known to cause many biologic effects, which are enhanced by the administration of interferon prior to or immediately after interleukin-2 infusion. Some of these effects could be related to the clinical response. METHODS: Sixteen patients with metastatic renal cell carcinoma were treated with continuous infusion of interleukin-2 plus alpha-2 interferon. Differential leukocyte count and lymphocyte subset evaluation were performed every 3 days during interleukin-2 treatment. At each cycle, the presence of the following antibodies was tested: antithyroid, antinuclear, antiplatelet and antierythrocyte. RESULTS: Fifteen patients were evaluable for response. No complete response was observed. Five patients obtained partial response (33%) and 3 stable disease (20%): 2 of them underwent surgical resection of metastases and obtained complete response. Some of our patients showed a significant increase in eosinophils, CD25+ lymphocytes and antithyroid antibodies. The association of these parameters, calculated with a "score" system, was also related to a better clinical response. CONCLUSIONS: Eosinophils, CD25+ lymphocytes and antithyroid antibodies could have a predictive value for the efficacy of interleukin-2 and alpha-2 interferon therapy in metastatic renal cell carcinoma.

Interleukin-2, interferon-alpha and interleukin-2 plus interferon-alpha in renal cell carcinoma. A randomized phase II trial.

BACKGROUND: The purpose of the present study was to investigate the therapeutic effectiveness of interleukin-2 (IL-2) and interferon (IFN), either alone or in combination, in comparable groups of patients affected by advanced renal cell carcinoma (RCC). PATIENTS AND METHODS: In order to limit selection biases, treatment was allocated on a random basis. Patients randomized to IL-2 alone were scheduled to receive eight rlL-2 24-hour i.v. infusion cycles, days 1 to 4, at a daily dose of 18 x 10(6) lU/m2 for a total of 25 weeks. Patients randomized to IFN alone were scheduled to receive rIFN-alpha at a daily dose of 6 x 10(6) IU/m2, days 1, 3 and 5, every week for a total of 52 weeks. Patients randomized to the combination of IFN and IL-2 were given the same drugs at the same daily doses for a total of 24 weeks. Drug dose was modified according to toxicity. RESULTS: Twenty-three percent (95% CI:+/-17.5) of patients treated with IL-2 alone showed an objective response to treatment (9% CR). The corresponding figures in patients treated with IFN alone or IFN plus IL-2 were 9% (95% CI:+/-11.9) and 9% (95% CI:+/-11.9), respectively. Complete responses were observed only in patients treated with IL-2. The median duration of response in the IL-2 arm was 18 months (range, 9.5-24). The duration of the two responses achieved by IFN alone was seven and nine months, respectively. The corresponding figures in the two patients responding to the combination of IFN with IL-2 were 19 and 27 months, respectively. Total IL-2 dose appeared to be a major predictor of response. Only a minority of patients experienced grade 3-4 toxicity, the incidence being higher in those treated with IL-2 or IL-2 plus IFN. CONCLUSIONS: Neither IFN nor IL-2 or the combination of the two appear to be very active in patients with advanced RCC, even when trial entry was restricted to patients with relatively indolent disease. This stresses the need for the development of new approaches.